Heme peroxidases comprise a large, structurally and catalytically diverse enzyme superfamily with representative members found throughout all kingdoms of life. These enzymes play important roles in many cellular processes, including stress-induced responses, defense mechanisms, and biosynthesis and degradation of various biological compounds. Heme peroxidases utilize peroxide to catalyze the one-electron oxidation of their respective substrates, and their catalytic mechanism is driven by the oxidation-reduction cycle of the heme-iron center. Despite conservation in general mechanism, the enzymes differ in the strength of their reduction potential, and correspondingly, in the degree of oxidative recalcitrance of the substrates that they can target. Some of the most catalytically powerful peroxidases, in terms of reduction potential, are produced and secreted by white rot basidiomycetes fungi to aid in the oxidative degradation of lignin (Hammel & Cullen, Curr Opin Plant Biol 11:349-355, 2008). Lignin is particularly difficult to oxidize owing to the complexity of its aromatic constituents and the diversity of linkages found throughout its structure, thus to oxidize lignin, peroxidases must generate a strong reduction potential. These high-reduction potential heme peroxidases are attractive candidates to meet the present and future needs of bio-based industry because their catalytic strength translates into an expanded substrate range, allowing them to oxidize general peroxidase targets, such as phenolic and aromatic amines, as well as more challenging substrates, such as lignin polymer models and non-phenolic aromatics (Watanabe et al., Struct Biol 169:226-242, 2010; Hammel & Cullen, supra).
Of the high reduction potential peroxidases, versatile peroxidase (VP), is of particular industrial interest because it has the catalytic versatility to directly oxidize a range of low- and high-reduction potential aromatic substrates and indirectly oxidize these substrates by oxidizing manganese, which acts as a diffusible oxidizer (Camarero, et al., J Biol Chem 274:10324-10330, 1999; Knop et al., Appl Environ Microbiol 82:4070-4080, 2016; Perez-Boada, et al., J. Mol. Biol 354:385-402, 2005). Among the heme peroxidase superfamily, VP is evolutionarily related to secretory plant and extracellular fungal heme peroxidases, and consequently, VP shares significant structural homology with these enzymes (Zamocky, et al., Arch Biochem Biophys 574:108-119, 2015). These peroxidases are predominantly α-helical in structure with two calcium coordination sites and numerous disulfide bridges to aid in structure stabilization (Watanabe, et al., supra; Ruiz-Duenas, et al., J Exp Bot 60:441-452, 2009). Key to their catalytic function, these enzymes also contain an internally coordinated heme molecule and oxidation pathways that lead from the enzyme surface to the heme center, facilitating heme access and in some cases, long-range electron transfer (LRET) (Ruiz-Duenas et al., supra; Smith & Veitch, Curr Opin Chem Biol 2:269-278, 1998. VP owes its catalytic versatility to the presence of three different oxidation pathways: (1) a manganese oxidation site; (2) an exposed heme edge, also found in general peroxidases that can directly oxidize phenols, amines, and small dye compounds; and (3) an LRET pathway capable of direct and indirect high-reduction potential aromatic oxidation (Knop et al., supra).
The reaction mechanism of VP can follow one of two mechanistic cycles (FIG. 1). The first of which is very similar to that of general peroxidases and enables VP to directly target low-reduction potential substrates and indirectly target high-reduction potential substrates through the oxidation of diffusible manganese (Hammel & Kullen, supra; Perez-Boada, et al., J Mol Biol 354:385-402, 2005). Alternatively, the addition of hydrogen peroxide can also lead to the activation of VP's LRET oxidation pathway, allowing the enzyme to directly target high-reduction potential and bulky substrates (Hammel & Cullen, Perez-Boada et al., both supra). The presence of this oxidation pathway facilitates VP's high-reduction potential and is one factor in differentiating its catalytic ability from that of general peroxidases. Another important factor in the high-reduction potential of VP is that its heme-iron is more electron-deficient than is found in general peroxidases, enabling VP to act as a stronger oxidant (Hammel & Cullen, Perez-Boada et al., both supra; Millis CD, Cai et al., Biochemistry 28:8484-8489, 1989). The electron-deficiency of the heme-iron is likely largely due to the residue composition of VP's heme coordination pocket and is predominantly influenced by the strength of the hydrogen bond it shares with a proximal histidine residue, resulting in a pentacoordinate ferric state (Carmarero et al., Perez-Boada et al., both supra; Banci et al., Proc Natl Acad Sci USA 88:6956-6960, 1991).
A major limiting factor in the industrial application of VP is that, despite its high oxidative power and substrate versatility, it functions under a fairly narrow range of in vitro conditions and has a relatively short half-life at its optimal pH (pH 3-4) (Saez-Jimenez, et al. PLoS One 10:e0140984, 2015). Thus, the range of VP's application and cost efficiency of its use is greatly diminished (Ayala, et al., J Mol Microbiol Biotechnol 15:172-180, 2008; Martinez, et al., Curr Opin Biotechnol 20:348-357, 2009). Heme peroxidase pH and temperature tolerance is associated with general structural stability, and a variety of structural factors have been implicated in the ability of heme peroxidases to tolerate a wide range of temperature and pH conditions. These key structural factors include: the presence of disulfide bridges and calcium coordination sites; surface charge and pI; total α-helical content and the presence of an additional α-helix in a variable loop region; and total number of proline residues and positioning of these prolines (Fernandez-Fueyo, et al Biotechnol Biofuels 7:2, 2014). Although VP and its structural homologs share a similar overall fold, they vary greatly in sequence identity, which translates into substantial differences in residue composition of the enzyme surface and heme coordination pocket as well as differences in precise disulfide bridge placement. Interestingly, one of the most temperature and pH tolerant heme peroxidases was identified from the leaves of the royal palm tree Roystonea regia (Sakharov, et al., Plant Sci 161:853-86, 2001). Royal palm tree peroxidase (RPTP) is a member of the plant heme peroxidase superfamily and structural homolog of VP, but it displays thermal stability on par with that of thermophilic microbial enzymes over a broad pH range (pH 2.8-10.3) (Watanabe et al. supra; Zamorano, et al., Biochimie 90:1737-1749, 2008).
Structural stability and its effect on temperature and pH tolerance is likely due to a combination of factors, rather than a single factor alone, and thus engineering efforts to enhance the temperature and pH tolerance of VP to improve the feasibility of its industrial use requires consideration of many different structural aspects. There have been several engineering endeavors directed at improving the pH stability of VP through directed evolution (Saez-Jimenez, et al., Biochem J 473:1917-1928, 2016) and rational design through mutagenesis of surface exposed residues (Saez-Jimenez et al., supra; Fernandez-Fueyo, et al., Biotechnol Biofuels 7:114, 2014). These engineering methods predominantly sought to leave VP's catalytic components untouched, and thus, focused on residue substitutions far from the oxidation pathways. In contrast to these previous methods, we describe a structure-guided approach to the rational design of a peroxidase with the high-reduction potential and substrate range of a VP but with enhanced temperature and pH tolerance of RPTP. To achieve this, we drew on the wealth of biochemical and structural information available on VP and RPTP, utilizing RPTP as a structural scaffold into which we built the catalytic components (oxidation pathways and heme coordination pocket architecture) of VP. This engineered peroxidase, VP2.0, combines the unique properties of these two enzymes—the catalytic versatility to oxidize manganese and low- and high-reduction potential substrates and an enhanced temperature and pH tolerance to outperform its catalytic parent over time.